EP2342332A1 - Mesure par spectroscopie d impédance de l adn - Google Patents

Mesure par spectroscopie d impédance de l adn

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Publication number
EP2342332A1
EP2342332A1 EP09813177A EP09813177A EP2342332A1 EP 2342332 A1 EP2342332 A1 EP 2342332A1 EP 09813177 A EP09813177 A EP 09813177A EP 09813177 A EP09813177 A EP 09813177A EP 2342332 A1 EP2342332 A1 EP 2342332A1
Authority
EP
European Patent Office
Prior art keywords
dna
transducer
electrode
electrode surfaces
immobilized probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09813177A
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German (de)
English (en)
Other versions
EP2342332A4 (fr
Inventor
Andrei L. Gindilis
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Sharp Corp
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Sharp Corp
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Filing date
Publication date
Application filed by Sharp Corp filed Critical Sharp Corp
Publication of EP2342332A1 publication Critical patent/EP2342332A1/fr
Publication of EP2342332A4 publication Critical patent/EP2342332A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • This invention generally relates to Deoxyribonucleic acid (DNA) detection and, more particularly, to a system and method for the measurement of DNA by impedance spectroscopy.
  • DNA Deoxyribonucleic acid
  • Microarray technology is a power research tool that permits the assaying of multiple analytes in a single sample - a multiplexed assay format.
  • a microarray has to contain multiple transducers modified with different bio-components. Selective attachment of a desired bio-component to a particular transducer constitutes one of the biggest challenges in the microarray technology.
  • the maj or approaches for microarray multiplexing are: (i) spotting of different bio- components over an array; (ii) physical separation of transducers via a nano-fluidic set of connections, and target delivery of bio-components to pre-selected transducers; (iii) self-assembling of tagged bio-components on an array surface that is modified with an agent capable of the specific capturing of bio-component tags; and (iv) controlled synthesis of bio-components on the surface of transducers.
  • PCR Real time polymerase chain reaction
  • PCR derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece . PCR can be extensively modified to perform a wide array of genetic manipulations.
  • PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus .
  • This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis .
  • DNA oligonucleotides also called DNA primers
  • the vast majority of PCR methods use thermal cycling, i. e. alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) b ⁇ the DNA polymerase to selectively amplify the target DNA.
  • the selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
  • a basic PCR set up requires several components and reagents. These components include:
  • a DNA polymerase such as Taq polymerase or another
  • DNA polymerase with a temperature optimum at around 70 0 C .
  • dNTPs Deoxynucleoside triphosphates
  • Buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase .
  • Fig. 1 is a schematic diagram depicting a PCR cycle (prior art) .
  • stage one 1) denaturing occurs, e.g. , at 94- 96°C for 20-30 seconds, melting the DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
  • stage two (2) annealing occurs, e . g. , at -65 0 C for 20-40 seconds, which permits the allowing annealing of the primers to the single-stranded DNA template.
  • the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used.
  • DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence.
  • the polymerase binds to the primer-template hybrid and begins DNA synthesis. In stage three (3) extension or elongation occurs, e . g. , at 72°C .
  • the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5 ' to 3' direction, condensing the 5' ⁇ phos ⁇ hate group of the dNTPs with the 3 '-hydroxyl group at the end of the nascent (extending) DNA strand.
  • the extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified.
  • the DNA polymerase will polymerize a thousand bases per minute .
  • the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
  • the solid lines represent the DNA template to which primers anneal that are extended by the DNA polymerase (circles) , to give shorter DNA products (hatched lines) , which themselves are used as templates as PCR progresses.
  • the PCR usually consists of a series of 20 to 40 repeated temperature changes called c ⁇ cles; each cycle typically consists of 2-3 discrete temperature steps . Most commonly, PCR is carried out with cycles that have three temperature steps. The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C) , and followed by one hold at the end for final product extension or brief storage.
  • the temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.
  • Quantitative PCR is used to measure the quantity of a PCR product (preferably real-time) . It is the conventional method used to quantitatively measure the starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The conventional method with the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR is a more appropriate contraction. RT-PCR commonly refers to reverse transcription PCR, which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.
  • fluorescent dyes such as Sybr Green
  • fluorophore-containing DNA probes such as TaqMan
  • Detection is based on differences in the fluorescence of dyes bound to single-strand versus double-strand DNA molecules.
  • the optically detectable dyes act as a label.
  • the practical application of fluorescence detecting techniques are limited by (i) the high initial expenditure of purchasing a PCR unit integrated with a fluorescent reader; (ii) the high ownership costs due to expensive consumables (fluorescent dyes and special kits) ; and, (iii) expensive maintenance of optical light-detecting equipment.
  • miniaturization of the fluorescent based real time PCR if possible, is very challenging.
  • Ferrocene based labels an organometallic compound Fe(CsHSh)
  • Fluorescent dyes and Ferrocene are both additives (labels) that unfortunately create background "noise” in the solution that can interfere with DNA measurement.
  • the application of labeled components in real time PCR has two major drawbacks: (i) insufficient stability of the label itself during thermal cycling process, and (ii) PCR bias associated with amplification error and chain reaction termination due to usage of a 'foreign' component. It is very desirable to avoid an application of any labeled components in the PCR process.
  • the DNA measurement approach described herein has a high potential for miniaturization and low-cost formats.
  • the principle of action is based on the coupling of a standard thermal cycling PCR process with a transducer capable of quantitative real time label-free detection of DNA products.
  • the transducer is based on an impedance spectroscopy electrode with specific surface functionalization.
  • an impedance spectroscopy method for quantitatively measuring Deoxyribonucleic acid (DNA) .
  • the method provides a transducer having electrode surfaces exposed to a shared environment (preferably, shared local environment) .
  • the electrode surfaces are functionalized with an oligonucleotide to interact with a predetermined DNA target.
  • a DNA sample solution is introduced into the environment.
  • the solution includes nucleotides, polymerase enzyme, and primers .
  • the DNA sample is thermocycled to promote a first DNA target polymerase chain reaction (PCR) .
  • PCR DNA target polymerase chain reaction
  • capacitance is measured between a pair of transducer electrodes, and in response to measuring the capacitance, a determination is made of the presence of first DNA amplicons in the DNA sample.
  • a number of thermocycles are performed and capacitance measurements are made after each cycle, so that an amplicon growth rate can be determined.
  • providing the transducer having electrode surfaces functionalized with the oligonucleotide includes either providing an immobilized probe molecule with a 3 ' end attached to the electrodes surfaces and a solution-exposed 5' end, or an immobilized probe molecule with a 5 ' end attached to the electrode surfaces and a solution-exposed 3 ' end. Additional details of the above described method and a system for selectively functionalizing a transducer microarray are provided below.
  • Fig. 1 is a schematic diagram depicting a PCR cycle
  • Figs . 2A and 2B are plan and partial cross-sectional views, respectively, depicting a microarray of transducers for the impedance spectroscopy quantitative measurement of DNA.
  • Fig. 3 is a schematic block diagram depicting a variation of the microarray of Fig. 2.
  • Figs . 4A and 4B are partial cross-sectional views depicting a transducer electrode surface in greater detail.
  • Figs . 5A and 5B are diagrams depicting cycles in a PCR process using a microarray with electrode surfaces functionalized as in Figs . 4A and 4B, respectively.
  • Fig. 6 is a graph depicting the relationship between the transducer response and the number of PCR cycles.
  • Fig. 7 is a flowchart illustrating an impedance spectroscopy method for quantitatively measuring DNA.
  • Fig. 8 is a graph depicting the impedance spectroscopy detection of a single stranded PCR product.
  • a transducer is a detector capable of generating a physical signal (output) in response to alterations of biological or chemical environment in the vicinity of the transducer's surface.
  • the signal may be visual or electrical, for example . These alterations occur when a pre-selected biological component that is attached to the transducer's surface specifically interacts with a target analyte, which is the process of bio-recognition.
  • the transducer integrated with the biological component forms a sensing element of a biosensor.
  • the biological component responsible for bio- recognition is a molecule capable of specific binding with the target analyte, specific transformation of the corresponding target analyte, or both.
  • An analyte is a substance or chemical constituent that is determined in an analytical procedure, such as a titration.
  • the analyte may be the ligand or the binder, while in blood glucose testing, the analyte is glucose.
  • analyte In medicine, the term “analyte” often refers to the type of test being run on a patient, as the test is usually determining a chemical substance in the human body. An analyte cannot typically be measured, but a measurable property of the analyte can be . For instance , glucose cannot be measured, but glucose concentration can be measured. In this example “glucose” is the component and “concentration” is the property. In laboratory and layman jargon the "property” is often left out provided the omission does not lead to an ambiguity of what property is measured.
  • Biotin and streptavidin can also be considered as biological components.
  • the transducer surface can be conductive, semi- conductive, or non-conductive .
  • the surface material can be metal or alloy such as gold, platinum, aluminum, chrome, or silica, carbon-based such as graphite or glassy carbon, glass, ceramic, a composite such as silicon nitride or indium tin oxide (ITO) , or a plastic such as polystyrene or nylon.
  • Transducer Surface Modification The initial modification of the transducer surface introduces functional groups that are capable of binding other biological components to the sensing element.
  • the introduction of the functional groups to the transducer surface can be performed in one of the following ways: - Direct chemical conversion of the transducer surface.
  • a carbon-based surface can be oxidized using oxygen plasma or an oxidant such as nitric acid.
  • the polymer can be a biopolymer (poly- sugar, gelatin, etc) , polyethyleneimine, polyacrylic acid, hydro-gel (polyvinyl alcohol, silica gel, etc.) , nylon, etc.
  • Functionalization describes the modification of a transducer surface with attached bio-probe molecules capable of specific biological recognition of analyte molecules. Biological recognition is an ability of the bio- probe molecule to specifically bind or catalytically convert analyte molecules.
  • Figs. 2A and 2B are plan and partial cross-sectional views, respectively, depicting a microarray of transducers for the impedance spectroscopy quantitative measurement of DNA.
  • Each transducer 202 has electrodes 204 and 206 with surfaces 208 exposed to a shared DNA sample environment solution 210. As explained in more detail below, the electrode surfaces 208 are functionalized with an oligonucleotide to interact with a predetermined first DNA target.
  • a measurement port 212 has electrical interfaces connected to measure capacitance between the electrodes 204 /206 of each transducer 202. As shown, the measurement port 212 includes an independent electrical interface for each electrode .
  • Lines 214a and 216a can be used to measure the capacitance between electrodes 204a and 206a.
  • Lines 214b and 2 16b can be used to measure the capacitance between electrodes 204b and 206b .
  • Lines 214n and 2 16n can be used to measure the capacitance between electrodes 204n and 206n.
  • a substrate 218 underlies the transducers 202 and a cover 220 overlies the transducers .
  • the combination of the substrate 2 18 and cover 220 forms a cavity 222 to provide the shared DNA sample environment solution 2 10.
  • Fig. 3 is a schematic block diagram depicting a variation of the microarray of Fig. 2.
  • the first electrode 204 of each transducer 202 is connected.
  • the measurement port 212 has a first electrical interface 300 connected to each first electrode 204 , and an independent electrical interface for each second electrode
  • Lines 300 and 216a can be used to measure the capacitance between electrodes 204 and 206a
  • Lines 300 and 216b can be used to measure the capacitance between electrodes 204 and 206b
  • Lines 300 and 216n can be used to measure the capacitance between electrodes 204 and 206n (transducer 202n) .
  • each transducer 202 includes a first electrode 204 formed in an interdigital pattern with respect to a second electrode 206.
  • the microarray is not limited to any particular electrode shape .
  • Figs . 4A and 4 B are partial cross-sectional views depicting a transducer electrode surface in greater detail.
  • the transducer electrode surface 208 in Fig. 4A is functionalized with an olignucleotide immobilized probe molecule 400 with a 3 ' end 402 attached to the electrodes surface 208 and a solution-exposed 5 ' end 404.
  • the immobilized probe molecule 400 is capable of binding to single stranded first DNA amplicons .
  • the transducer electrode surface 208 is functionalized with an olignucleotide immobilized probe molecule 406 with a 5' end 408 attached to the electrode surface 208 and a solution-exposed 3' end 4 10.
  • the immobilized probe 406 is capable of binding to single stranded first DNA amplicons, and acting as a primer to enzynmatically extend antisense single stranded first DNA amplicons from the immobilized probe molecules.
  • the PCR thermal cycling process is based on a polymerase extension reaction that involves a target DNA molecule and two specific primers.
  • the process is an exponential amplification, and thus, results in formation of a large amount of copies of the target DNA sequence located between the primers .
  • Figs. 5A and 5B are diagrams depicting cycles in a PCR process using a microarray with electrode surfaces functionalized as in Figs. 4A and 4B , respectively.
  • the transducer surface 208 is modified with a probe capable of specific hybridization with one of the strands of DNA template .
  • the transducer can be modified with one of the primers attached to the surface via its 5' end.
  • the double-strand DNA molecule denatures forming two single strands .
  • the single strands compete for binding with the free primers in solution, and the probe on the transducer surface .
  • a fraction of the DNA strands binds to the transducer surface
  • Fig. 5A resulting in an increase of the transducer response.
  • the probe on the transducer surface is a specific primer (Fig. 5B)
  • Fig. 5B the target molecule binding to the surface is followed by enzymatic extension of the probe on the surface. This process causes an increase in the size of molecules attached to the transducer surface and leads to a transducer response .
  • the attachment of additional molecules to the transducer surface is proportional to the amount of amplified DNA product present in the reaction mixture .
  • the transducer response permits a real-time quantification of DNA concentration in the sample during PCR cycling.
  • Fig. 6 is a graph depicting the relationship between the transducer response and the number of PCR cycles. The relationship is a function of the initial concentration of DNA template in the sample, and can be used for quantitative determination of the DNA template in a way similar to the conventional fluorescent real time PCR (see for example (A) The Journal of Physiology, July 1 , 2006 574, 229-243 , (B) BioRad (Hercules, CA) protocols : http: / /www3.bio- rad.com/ gexp / html/ support / amp_central/ ac203c.html, (C) AMRESCO (Solon, OH) protocols : http : / / www. amresco- inc.
  • Fig. 7 is a flowchart illustrating an impedance spectroscopy method for quantitatively measuring DNA. Although the method is depicted as a sequence of numbered steps for clarity, the numbering does not necessarily dictate the order of the steps . It should be understood that some of these steps may be skipped, performed in parallel, or performed without the requirement of maintaining a strict order of sequence.
  • the method starts at Step 700.
  • Step 702 provides a transducer having electrode surfaces exposed to a shared local environment.
  • the electrode surfaces are functionalized with an oligonucleotide to interact with a predetermined first DNA target.
  • Step 704 introduces a DNA sample solution, including nucleotides, polymerase enzyme, and primers, into the local environment.
  • Step 706 thermocycles the DNA sample to promote a first DNA target polymerase chain reaction (PCR) .
  • Step 708 measures capacitance or impedance between a pair of transducer electrodes.
  • Step 710 determines the presence of first DNA amplicons in the DNA sample .
  • Step 708 includes measuring capacitance in a plurality of thermocycles, and comparing the plurality of capacitance measurements.
  • Step 702 includes providing either an olignucleotide immobilized probe molecule with a 3' end attached to the electrodes surfaces and a solution-exposed 5 ' end (Fig. 4A) , or an olignucleotide immobilized probe molecule with a 5' end attached to the electrode surfaces and a solution- exposed 3' end (Fig. 4B) .
  • Step 702 provides a first electrode formed in an interdigital pattern with respect to a second electrode.
  • thermocycling the DNA sample to promote the first DNA target PCR in Step 706 includes substeps, in each thermocycle.
  • Step 706a denatures the first DNA sample at a first temperature
  • Step 706b anneals the first DNA sample at a second temperature, lower than the first temperature .
  • Step 706c performs an extension stage, after annealing, at a third temperature in a range between the first and second temperatures.
  • thermocycling the DNA sample in Step 706 may include performing 20 to 50 thermocycles.
  • the denaturing performed in Step 706a may be performed at a temperature of about 95 0 C
  • Step 706b may anneal at a temperature in the range of about 45 to 75 0 C .
  • Step 702 provides electrode surfaces with an olignucleotide immobilized probe molecule with a 3 ' end attached to the electrodes surfaces and a solution-exposed
  • thermocycling the DNA sample in Step 706 includes binding single stranded first DNA amplicons to the immobilized probe molecule in response to each cycle of annealing. Then, measuring capacitance between the pair of transducer electrodes in Step 708 includes measuring capacitance following each cycle of annealing.
  • thermocycling the DNA sample includes sustaining a bond between single stranded first DNA amplicons and the immobilized probe molecule following each cycle of denaturing.
  • measuring capacitance in Step 708 includes measuring capacitance following each cycle of denaturing.
  • thermocycling the DNA sample in Step 706 includes binding single stranded first DNA amplicons to the immobilized probe molecule .
  • the probe acts as a primer to enzynmatically extend antisense single stranded first DNA amplicons from the immobilized probe molecules in response to each extension stage. Then,
  • Step 708 may measure capacitance after each stage of extension.
  • Fig. 8 is a graph depicting the impedance spectroscopy detection of a single stranded PCR product. The measurement was performed to detect a long DNA fragment obtained as a PCR product. (Experiment)
  • a bacterial genome was amplified by PCR to form a double stranded DNA as follows (Sequence ID No . l ) : AATATGGTATTCCGCAAATCTCCACTGGCGATATGCTGCGTGCT GCGGTCAAATCTGGCTCCGAGCTGGGTAAACAAGCAAAAGACA
  • Impedance spectroscopy test was performed at 150 mV.
  • the cell transducer electrode surface
  • PBST was removed from the cell and the sample was added.
  • the results show an impedance (Z) dependence on time .
  • the addition of the DNA-containing sample resulted in increase of impedance at a 20 Hz scan frequency.
  • the impedance increase tends to saturate after approximately 1000 seconds . This saturation corresponds to a complete binding of the target DNA to a probe functionalized surface of the interdigitated electrode.

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Abstract

La présente invention concerne un système et un procédé de spectroscopie d’impédance destinés à mesurer quantitativement l’ADN. Le procédé fournit un transducteur ayant des surfaces d’électrodes exposées à un environnement local partagé. Les surfaces d’électrodes sont fonctionnalisées avec un oligonucléotide afin d’interagir avec une cible ADN prédéterminée. Une solution échantillon d’ADN est introduite à l’intérieur de l’environnement local. La solution comprend des nucléotides, une enzyme polymérase, et des amorces. L’échantillon d’ADN est thermocyclé afin de favoriser une première réaction en chaîne par polymérase (PCR) de la cible ADN. Ensuite, une capacité électrique est mesurée entre une paire d’électrodes du transducteur, et en réponse à la mesure de la capacité électrique, une détermination est effectuée en présence des premiers amplicons d’ADN dans l’échantillon d’ADN. Typiquement, un nombre de thermocycles est effectué et des mesures de la capacité électrique sont effectuées après chaque cycle, de sorte qu’une vitesse de croissance de l’amplicon peut être déterminée.
EP09813177A 2008-09-12 2009-09-09 Mesure par spectroscopie d impédance de l adn Withdrawn EP2342332A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US12/210,062 US20100069253A1 (en) 2008-09-12 2008-09-12 Impedance Spectroscopy Measurement of DNA
PCT/JP2009/066059 WO2010030035A1 (fr) 2008-09-12 2009-09-09 Mesure par spectroscopie d’impédance de l’adn

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EP2342332A1 true EP2342332A1 (fr) 2011-07-13
EP2342332A4 EP2342332A4 (fr) 2012-08-22

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